A common application of tunable sources of femtosecond laser-pulses (laser-radiation pulses having a pulse-duration from a few femtoseconds to a few tens of femtoseconds) is in multi-photon excitation (MPE) of analysis samples in microscopy and spectrometry. The most commonly used such sources are so-called ultrafast lasers employing solid-state gain-media with a relatively very broad gain-bandwidth. The most common such gain medium is titanium-doped sapphire (Ti:sapphire), which has a FWHM gain-bandwidth extending from about 700 nanometers (nm) to about 900 nm. Laser oscillation can be achieved over a wavelength range between about 650 nm and 1080 nm, but with noticeably lower power, for example less than 1 Watt (W) at wavelengths greater than 1000 nm and less than 700 nm. There are other broad-band gain-media operable in about the same wavelength range, such as alexandrite, chromium-doped lithium strontium aluminum fluoride (Cr:LISAF), and chromium-doped lithium calcium aluminum fluoride (Cr:LICAF). The term “vibronic” gain-media is often used by practitioners of the art for such broad-band gain-media.
Ultrafast laser sources employing vibronic gain-media are relatively expensive compared with other pulsed solid-state lasers of equivalent power. This is due to the fact that the gain media are pumped by expensive visible (frequency converted) CW lasers. In addition, the wide tuning range requires complicated resonator designs to control the emission wavelength and compensate for group-delay dispersion.
Pulse wavelengths suitable for MPE are not restricted to the 700-nm to 900-nm (FWHM) wavelength range of the vibronic gain-media, but can extend further into the near infrared (NIR) region of the electromagnetic spectrum, where several fluorophores (fluorescent markers) can be excited by two-photon (2P) absorption. These include green fluorescent protein, such as EGFP; red fluorescent protein, such as mCherry; chimeric opsins, such as channel rhodopsin ChR2, and C1V1; and calcium signal fluorophores, such as GCaMP3 and RCaMP1. Suitable 2-photon excitation wavelengths range from 900 nm for EGFP to 1110 nm for RCaMP. In each case, the absorption band of the fluorophore is sufficiently wide that there is a tolerance of about ±30 nm or greater around a nominal peak within which the fluorophore can be effectively excited.
In certain cases, it could be advantageous in an analysis if two or more fluorophores were used, and simultaneously excited by two or more corresponding wavelengths. The term “simultaneously”, here, meaning, for example, within the response time of a human eye or a CCD.
Pulses having wavelengths in the above-discussed NIR range can be provided (tunably) by using an ultrafast laser or MOPA to pump an optical parametric oscillator (OPO). This, however, adds another layer of cost and complexity to the laser. Further, only one of the wavelengths can be generated at any one time.
There is a need for a relatively inexpensive source of femtosecond laser pulses for MPE that can directly generate NIR laser pulses at different wavelengths. Preferably, the laser pulse-source should be capable of generating pulses at two or more NIR wavelengths simultaneously, with at least limited tunability, for selecting the wavelengths.